| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1 Pediatrics and 2 Physiology and 3 Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
We used the patch-clamp technique to study the
effect of nitric oxide (NO) on a cation channel in rat type II
pneumocytes [alveolar type II (AT II) cells].
Single-channel recordings from the apical surface of AT II cells in
primary culture showed a predominant cation channel with a conductance
of 20.6 ± 1.1 (SE) pS (n = 9 cell-attached patches) and
Na+-to-K+
selectivity of 0.97 ± 0.07 (n = 7 cell-attached patches). An NO donor,
S-nitrosoglutathione (GSNO; 100 µM),
inhibited the basal cation-channel activity by 43% [open
probability (Po), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03;
P < 0.001;
n = 16 cell-attached patches],
with no significant change in the conductance. GSNO reduced the
Po by reducing
channel mean open and increasing mean closed times. GSNO inhibition was
reversed by washout. The inhibitory effect of NO was confirmed by using
a second donor of NO,
S-nitroso-N-acetylpenicillamine (100 µM; Po,
control 0.53 ± 0.05 vs.
S-nitroso-N-acetylpenicillamine 0.31 ± 0.04;
42%; P < 0.05;
n = 5 cell-attached patches). The GSNO
effect was blocked by methylene blue (a blocker of guanylyl cyclase;
100 µM), suggesting a role for cGMP. The permeable analog of cGMP,
8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner
similar to GSNO
(Po, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02;
P < 0.05;
n = 7 cell-attached patches).
Pretreatment of cells with 1 µM KT-5823 (a blocker of protein kinase
G) abolished the inhibitory effect of GSNO. The NO inhibition of
channels was not due to changes in cell viability. Intracellular cGMP
was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml;
P < 0.05;
n = 6 cell-attached patches). We
conclude that NO suppresses the activity of an
Na+-permeant cation channel on the
apical surface of AT II cells. This action appears to be mediated by a
cGMP-dependent protein kinase.
guanosine 3',5'-cyclic monophosphate; nonselective cation channel; alveolar type II cells; S-nitrosoglutathione; sodium channel; single-channel recording; amiloride
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
DISTAL LUNG EPITHELIUM plays a critical role in maintaining normal alveolar fluid balance (1, 9, 19, 20) and in the adaptation of newborn lungs to air breathing (9, 22). The alveolar walls, lined by type I and type II cells, regulate the fluid to keep the alveoli moist while avoiding excessive buildup of fluid. Transepithelial fluid movement appears largely to be a result of active salt transport, which drives the osmotic movement of water. Recent patch-clamp studies show that Na+ channels, located on the apical surface of alveolar type II (AT II) cells, allow vectorial transport of Na+ from the alveolar space into the cell, with subsequent extrusion into the interstitium by Na+-K+-ATPase located on the basolateral membrane (7, 17, 19, 20). The interstitial fluid is then taken up by the flow vessels and lymphatics. The exact mechanism by which the lung epithelial cells control fluid reabsorption and prevent pulmonary edema is not clear, although disruption of this process has been implicated in several disease states.
The use of inhaled nitric oxide (NO) is currently being evaluated in a variety of lung disorders including pulmonary hypertension (25), acute respiratory distress syndrome (27), and high-altitude pulmonary edema (28). Studies done in vitro and in vivo suggest that NO may have an effect on lung fluid dynamics, although the mechanism underlying the NO effect is largely unknown. Because alveolar epithelial cells are exposed to high concentrations of NO during inhaled NO treatment, it is possible that NO may alter lung epithelial Na+ and water transport. In the kidney, NO has been shown to inhibit Na+ reabsorption by cultured cortical collecting duct cells (29). In the lung, Compeau et al. (4) have shown that endotoxin-stimulated alveolar macrophages impair distal lung epithelial ion transport by inactivating amiloride-sensitive, nonselective cation (NSC) channels. This inhibition was dependent on NO synthesis by the macrophage, suggesting that NO may promote lung edema formation by inhibiting cation channels in the AT II cells. However, other investigators have shown that NO prevents pulmonary edema formation in the isolated rat lung (8) and in humans prone to high-altitude pulmonary edema (28). The reasons for the discrepancy in the findings of these investigators remain to be elucidated.
The objective of this study was to examine the effect of NO on lung epithelial Na+ transport and to determine its mechanism of action. We used the patch-clamp technique to study the effect of NO on an amiloride-sensitive, Na+-permeable cation channel on the apical surface of rat AT II cells. Our results show that NO inhibits these cation channels (and, presumably, Na+ reabsorption) by AT II cells and that this inhibition is mediated by intracellular cGMP acting through a cGMP-dependent protein kinase (PK).
| |
METHODS AND PROCEDURES |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Type II pneumocyte isolation and culture. AT II cells were isolated by enzymatic digestion of lung tissue from adult Sprague-Dawley rats (200-250 g) with published techniques (2). Briefly, the rats were anesthetized with pentobarbital sodium and heparinized (100 units/kg). AT II cells were digested by tracheal installation of elastase (0.4 mg/ml). Lung tissue was minced in DNase (1 mg/ml) and filtered sequentially through 100- and 20-µm nylon mesh. Purification was based on the differential adherence of cells to dishes coated with rat IgG. Nonadherent AT II cells were collected, centrifuged, and seeded onto glass coverslips (~2 × 105 cells/cm2) in Dulbecco's modified Eagle's medium-F-12 medium containing 5% FCS and antimicrobial agents and supplemented with L-glutamine and Na+ bicarbonate. Cells were incubated in 90% air-10% CO2 and used for patch-clamp studies between 24 and 96 h after harvest. No significant difference in Na+-channel activity was observed within this time frame. Cell viability (90%) and purity (95%) associated with this isolation procedure have been validated in our laboratory (2).
Solutions and drugs. All solutions were made with deionized water and then passed through a 0.2-µm filter (Gelman Sciences, Bedford, MA) before use. The bath and pipette solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 MgCl2, 1 CaCl2, 5 KCl, and 10 HEPES, pH 7.4 with 2 N NaOH. In the inside-out recordings, the pipette solution was the same, but the bath solution was changed to (in mM) 5 NaCl, 140 KCl, 4 CaCl2, 5 EGTA, 1 MgCl2, and 10 HEPES, pH 7.4 with 2 N KOH. The contents of the bathing and pipette solutions were varied as appropriate for specific protocols. All chemicals were obtained from Sigma (St. Louis, MO) except 8-bromo-cGMP (8-BrcGMP) and KT-5823 that were from Calbiochem.
Procedure for single-channel
recordings. Patch-clamp experiments were carried out at
room temperature. The pipettes were pulled from filamented borosilicate
glass capillaries (TW-150, World Precision) with a two-stage vertical
puller (Narishige, Tokyo, Japan). The pipettes were coated with Sylgard
(Dow Corning) and fire polished (Narishige). The resistance of these
pipettes was 5-8 M
when filled with pipette solution. We used
the cell-attached configuration for most of our studies because, in
this configuration, the cytoplasmic constituents remain intact, thus
allowing us to study the role of cytoplasmic second messengers in the
regulation of ion-channel activity. Inside-out patches were also used
to determine the selectivity of the channel and to determine whether the effects of agents were directly on the channel or mediated by a
signaling cascade. After formation of a high-resistance seal (>50
G) between the pipette and the cell membrane, channel currents were
sampled at 5 kHz with a patch-clamp amplifier (Axopatch 200A, Axon
Instruments, Foster City, CA) and filtered at 1 kHz with an eight-pole,
low-pass Bessel filter. Data were recorded by a computer with pCLAMP 6 software (Axon Instruments, Foster City, CA). Current-amplitude
histograms were made from stable continuously recorded data, and the
open and closed current levels were determined from least square fitted
Gaussian distributions. We used the product (NPo) of the
number of channels (N) times the
open probability (Po) as a
measure of the activity of the channels within a patch. This product
could be calculated from the single-channel record without making any
assumptions about the total N in a
patch or the Po
of a single channel
|
To determine whether changes in
NPo were due to a
change in Po, the
mean open (
open) and closed
(closed) times were
determined. open and
closed are experimental
measures that can provide information as to the average duration in all
open and closed states. The mean
open and
closed for
N observed channels can be calculated from the following equations
|
|
Procedure for cGMP estimations. Intracellular cGMP levels were measured in cultured AT II cells with an enzyme immunoassay (Biotra EIA, Amersham, Arlington Heights, IL). Briefly, cells were treated with S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and carbachol for 20 min, and the reaction was stopped by removal of the incubation medium and addition of 65% ice-cold ethyl alcohol. The intracellular cGMP extracted into the supernatant was measured by enzyme immunoassay in duplicate.
Methods for statistical analysis. Statistical analysis for the changes in the Po of channels and the biochemical estimations were performed with SPSS for Windows. Statistical significance between two groups was determined by paired or unpaired t-tests as appropriate. When the comparison between more than one group was required, statistical significance was usually determined by one-way ANOVA followed by pairwise comparisons with a Bonferroni t-test to determine significant differences between each group. A P value < 0.05 was regarded as significant. Because of variability in the mean open and closed times of control cells, the effects of GSNO were determined with a Kruskal-Wallis one-way ANOVA on ranks and Dunn's method to determine statistically significant differences from control values.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
All cells used for the present experiments had lamellar bodies and
other phenotypic features of AT II cells. The predominant Na+-permeant channel seen in
apical cell-attached patches is shown in Fig.
1A. This
channel had a linear current-voltage
(I-V)
relationship (Fig. 1B) with a
conductance of 20.6 ± 1.1 pS (n = 9 cell-attached patches) with 140 mM NaCl in the bath and pipette. No
rectification of the
I-V
curve was observed (Fig. 1B). The
pipette potential at which current polarity reversed was estimated to
be 37 mV. Because the resting membrane potential of alveolar
epithelium has previously been shown to be approximately 30 to
40 mV, the reversal potential appears to be close to 0 mV, which
would be expected for an NSC channel (20). Ion selectivity was
determined with inside-out recording and solutions of varying ionic
compositions. The channel had a similar permeability to
Na+ and
K+
(Na+-to-K+
permeability = 0.97 ± 0.07; n = 7 cell-attached patches). The channel
Po was decreased
by amiloride (0.1-1 µM) applied to the extracellular side (i.e.,
in the micropipette;
Po, control 0.31 ± 0.01 vs. amiloride 0.03 ± 0.01;
P < 0.01;
n = 7 cell-attached patches). More
than one current level was observed in 85.7% of active patches. Thus
this channel was very similar in characteristics to the NSC channel
described by Orser et al. (24) and Marunaka (17).
|
NO reduces the Po of apical NSC channels.
To investigate the acute effects of NO on NSC channel activity, we used
two agents that are known to release NO. GSNO (100 µM) was applied to
the bath solution after apical cell-attached recording was established.
The GSNO stock solution was freshly prepared just before
it was added to the bath. Figure
2A
shows the typical time course of NSC channel activity after exposure to
GSNO in the bath. With each cell-attached patch acting as its own
control, channel activity, measured as
Po, consistently
decreased from a mean control value of 0.28 ± 0.05 to a mean
treated value of 0.16 ± 0.03 (43%;
P < 0.001;
n = 16 cell-attached patches; Fig.
2B). The effect was immediate in
onset and was sustained for up to 30 min of recording in stable
patches. It was reversible, with a return to control levels after
washout (Po,
control 0.16 ± 0.05 vs. 100 µM GSNO 0.062 ± 0.03 vs. washout
0.21 ± 0.06; Fig. 2C). There was
no change in the conductance of the channel. An alternate donor of NO,
SNAP (100 µM), caused a decrease similar to that of GSNO in the
Po of the channel
(control 0.53 ± 0.05 vs. SNAP 0.31 ± 0.04; 42%;
P < 0.05;
n = 5 cell-attached patches; Fig.
3). This effect was not seen when GSH, the
carrier of NO in GSNO, was used in a 100 µM concentration
[Po,
control 0.35 ± 0.11 vs. GSH 0.27 ± 0.07;
P = not significant (NS);
n = 8 cell-attached patches;
Fig. 4]. Taken together,
these experiments indicate that NO released by the NO donors suppresses
basal NSC channel activity in apical cell-attached patches in AT II
cells.
|
|
|
|
|
76%;
P < 0.05;
n = 7 cell-attached patches; Fig.
8).
|
|
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Research during the past several years utilizing a variety of approaches has underscored the physiological importance of active Na+ transport by alveolar epithelium. Disruption of this process has been implicated in a number of disease states. Because several pharmacological agents, especially those applied topically to the lung epithelium, have the potential for altering epithelial ion and water transport, their effect on lung fluid balance warrants detailed study before they are inducted into the clinical armamentarium. The major findings of this study are that NO inhibits an NSC channel in AT II cells and that this action is mediated by a cGMP-activated PK. This is the first study to report the effect of NO on cation channels in the distal lung epithelia. We believe that this effect may be beneficial in some situations and may be harmful in others. A classic example of the former situation is cystic fibrosis in which increased Na+-channel activity results in viscid secretions because of excessive salt and water reabsorption from the alveolar spaces. Agents inhibiting Na+ transport by the lung epithelium would have a beneficial role in cystic fibrosis, and aerosolized amiloride has been employed with some benefit. Our observation that GSNO inhibits amiloride-sensitive Na+ transport in the lung points to a novel role for this compound with its potential bronchodilator, antimicrobial, and vasoregulatory properties. However, our study suggests that lung conditions accompanied by pulmonary edema could potentially be worsened by NO treatment. This is especially important if the lung edema is not associated with pulmonary hypertension. Because cation channels with characteristics similar to those reported in this paper have been reported from a variety of tissues, as has the ability to produce NO locally, our results could have a greater general significance.
Lung epithelial cation channels. A
complete understanding of the role of NSC channels in the physiology of
lung water has yet to be achieved. Our study examined a 20.6-pS NSC
channel recorded from apical cell-attached patches of AT II cells in
primary culture. When grown under the conditions described in
METHODS AND PROCEDURES, this was the
predominant cation-permeable channel in AT II cells. The presence of
NSC channels in alveolar epithelial cells has been shown by several
investigators (7, 18, 24). Orser et al. (24) studied fetal distal lung
epithelial cells from 20-day-gestation rat fetuses cultured on
collagen-coated coverslips. Using symmetrical solutions and inside-out
recording, the investigators observed single channels with a
conductance of 23 ± 1.1 pS and an
Na+-to-K+
permeability of 0.9. These channels were blocked by amiloride applied
to the apical side of the membrane. Marunaka (17) described an NSC
channel with a linear
I-V
relationship and a single-channel conductance of 26.9 ± 0.8 pS in
the fetal distal lung epithelium. Feng et al. (7) recently described a
similar NSC channel observed in apical cell-attached and inside-out
patches from rat AT II cells. Like the channels observed by us, these
channels are nonselective (Na+-to-K+
permeability = 1), voltage independent, and inhibited by amiloride. A
wide variety of single-channel properties has been reported for
amiloride-sensitive cation channels, including single-channel conductances ranging from 1 to over 50 pS (7, 11). It has been proposed
that different combinations of the various subunits comprising the
channel (
, 
, and 
) could produce channels with varying unitary
conductances (3, 11, 31). Kizer et al. (13) recently showed that
expression of the -subunit of the epithelial
Na+ channels from osteoblasts into
a null cell line (LM TK
)
resulted in an NSC channel
(Na+-to-K+
permeability = 1.1 ± 0.1) and a conductance of 24.2 ± 1.0 pS. Alternatively, the conductance could reflect the ionic conditions and
membrane composition in the tissue, which determine the physical state
of the membrane (11). We have also observed considerable variability in
the Po of these
channels. Such variabilty has also been observed in single-channel
recordings of amiloride-sensitive channels in cultured
Xenopus renal cells, human
lymphocytes, rat osteoclasts, and rat colonic epithelial cells. The
exact physiological role for these NSC channels is unclear, although
Tohda et al. (30) showed that these channels may play a role in the
increased reabsorption of fluid by alveolar epithelia in response to
-agonist stimulation.
Effect of NO on apical NSC channels. The inhibition of NSC channels by NO suggests that NO may play a role in the regulation of alveolar fluid and edema formation. The inhibitory effect of NO on NSC channels is consistent with studies by Stoos et al. (29), who showed that NO inhibits Na+ reabsorption in the isolated cortical collecting duct, and Koivisto and Nedergaard (14), who found that NO donors block NSC channel activity in rat brown adipose tissue. Compeau et al. (4) showed that endotoxin-stimulated alveolar macrophages impair distal lung epithelial ion transport by inactivating amiloride-sensitive NSC channels. These investigators showed a 60% reduction in amiloride-sensitive short-circuit current and a 60% decrease in the density of 25-pS NSC channels on the apical membrane of epithelium exposed to endotoxin and macrophages. This effect was blocked by NG-monomethyl-L-arginine, suggesting an NO effect. These studies are in contrast to studies by Guidot et al. (8), who used isolated perfused rat lungs to show that inhaled NO prevents a neutrophil-mediated, oxygen radical-dependent leak in isolated perfused rat lungs. The investigators reported a modest reduction in pulmonary arterial pressure 30 min after NO exposure but felt that NO prevented an oxygen radical-dependent leak in the lungs. The question of whether NO increases or decreases the propensity for pulmonary edema is yet to be resolved. It is possible that in in vivo studies where pulmonary hypertension is contributing to pulmonary edema formation, inhaled NO may act by reducing the hydrostatic pressure and hence alveolar fluid formation. In situations where pulmonary vasoconstriction is not a major player and in vitro, NO appears to worsen pulmonary edema by impeding epithelial ion transport. NO may also have an effect on Na+-K+-ATPase, but we have not addressed this in our study. The answer to these questions is important because inhaled NO is currently undergoing clinical trials in a variety of lung disorders.
The effects of GSNO and SNAP are generally attributed to the release of NO. The fact that two biochemically different but specific NO-releasing compounds, GSNO and SNAP, were equipotent in their effect on the NSC channel suggests a common mechanism of action. In this study, GSH (carrier of NO in GSNO) did not affect the NSC channels, suggesting that GSNO was acting via release of NO (3). We were able to demonstrate that the GSNO effect can be reversed by washout. One puzzling observation was the apparent stimulating effect of washout on patches previously exposed to GSNO. Possible mechanisms for this phenomenon may include suppression of endogenous NO and/or cGMP production by exogenous GSNO. Once the inhibitory effect of GSNO was washout, there was an increase in channel activity attributable to the lower level of endogenous inhibition.
The concentration of NO donors used in this study is higher than the range of NO concentrations encountered in the physiological state (10). However, because NO has a short half-life and needs to diffuse inside the cell for its action, the actual effective concentration of NO inside the cell may have been lower than the donor concentration used. Ichimori et al. (10) found that 100 µM SNAP generated a stable concentration of 0.1 µM NO at 25°C, a concentration that is well within the physiological range.
The interval histograms are consistent with a minimum model of the NSC channel that has one short-duration and one long-duration open state and two similar closed states. Such a model can be represented by the following kinetic scheme
|
NO acts via cGMP-dependent activation of a PK. Guanylate cyclase stimulation is believed to be responsible for many of the physiological and pathological effects of NO (21). We hypothesized that NO was acting on NSC channels via a guanylate cyclase-mediated increase in cGMP. We found that a permeable analog of cGMP (8-BrcGMP) had a virtually identical effect on NSC channels as did GSNO and SNAP. To further examine the role of cGMP in the inhibition of NSC channels, we utilized MeB to block soluble guanylate cyclase. We found that MeB abolished the effect of NO, suggesting that the inhibitory effect of GSNO on NSC channels in AT II cells is largely mediated by cGMP.
These studies show that NO acts on NSC channels via a cGMP-dependent mechanism. This is clear from the fact that cGMP analogs mimic and MeB blocks the action of NO. Furthermore, AT II cells respond to NO by production of cGMP. These findings are consistent with other studies (4, 5, 31) that have suggested a role for intracellular second messengers as modulators for ion-channel activity. Rocha and Kudo (26) showed that, in the kidney, hormones such as atrial natriuretic factor that increase cGMP levels result in inhibition of Na+ reabsorption. Light et al. (16) confirmed this with patch-clamp studies in which they showed that cGMP inhibits cation channels both directly and through a cGMP-dependent PK. We have also shown that cGMP acts via activation of PKG. It is possible that NO may have additional effects through the tyrosine kinase pathway, or the G protein-coupled receptor, via release of cytokines or other second messengers. The physiological regulation of epithelial Na+ channels appears to be complex because, in addition to the pathway discussed, channel activity is also modulated by methylation, arachidonic metabolites, and interactions with the cytoskeleton (15, 16).
There is considerable indirect evidence that cGMP action on renal epithelial Na+ channels is mediated via activation of PKG, leading to phosphorylation of cation channel or some related protein (5, 6). In the present study, we used KT-5823, a blocker of PKG (12), to study whether the NO-cGMP-induced inhibition of the channels was mediated by PKG. We found that KT-5823 blocked the effect of NO, suggesting a role for PKG in the observed effect of NO on the cation channels. It is possible that higher concentrations of KT-5823 may inhibit other PKs, which can then affect the channel under study or other related proteins (32).
Other mechanisms may contribute, in part, to the observed inhibition of NSC channels. These include a direct cGMP effect on the membrane and a phosphodiesterase (PDE)-mediated fall in cAMP levels because cAMP is known to stimulate epithelial Na+ channels (23). To invoke this mechanism, one would assume that elevated cGMP levels lead to an increase in PDE concentration that then lowers the cellular cAMP concentration. Whether there is a role for PDE in the NO-mediated inhibition of NSC channels is not clear.
Exposure of AT II cells to NO does not cause cell death. It is possible that at the concentrations used in this study, NO may be toxic to epithelial cells. We did not find any difference in the viability of the cells after exposure of AT II cells to be NO donors under the conditions used in our patch-clamp protocols. Furthermore, reversibility of NO effect after washout confirms that the inhibition of NSC channels was not related to any permanent changes in the cell and/or due to spontaneous "rundown" of channels in the patches being examined.
In summary, our study suggests that NO inhibits cation channels on the apical surface of AT II cells via a cGMP-mediated action. This suggests that NO may have a regulatory role in lung epithelial Na+ transport. We speculate that pharmacological modalities, which act via this mechanism, may affect lung Na+ and water transport. Although NO or its donors may have a therapeutic role in patients with cystic fibrosis, patients with preexisting lung edema need to be closely monitored for worsening of edema when being treated with inhaled NO.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to B. Reynolds for assistance with preparation of this manuscript.
| |
FOOTNOTES |
|---|
Support was provided by American Lung Association Award RG-133-N (to L. Jain) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37963 (to D. C. Eaton).
Preliminary results were presented at the Society for Pediatric Research meeting in Washington, DC, in May 1997 and the American Thoracic Society meeting in San Francisco, CA, in May 1997.
Address for reprint requests: L. Jain, Dept. of Pediatrics, Emory Univ. School of Medicine, 2040 Ridgewood Dr., NE, Atlanta, GA 30322.
Received 14 April 1997; accepted in final form 11 December 1997.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
1.
Bland, R. D.,
and
C. A. Boyd.
Cation transport in lung epithelial cells derived from fetal, newborn, and adult rabbits.
J. Appl. Physiol.
61:
507-515,
1986
2.
Brown, L. A. S.,
C. Bai,
and
D. P. Jones.
Glutathione protection in alveolar type II cells from fetal and neonatal rabbits.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L305-L312,
1992
3.
Canessa, C. M.,
L. Schild,
G. Buell,
B. Thorens,
I. Gautschi,
J. D. Horisberger,
and
B. C. Rossier.
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
Nature
367:
463-467,
1994[Medline].
4.
Compeau, C. G.,
O. D. Rotstein,
H. Tohda,
Y. Marunaka,
B. Rafii,
A. S. Slutsky,
and
H. O'Brodovich.
Endotoxin-stimulated alveolar macrophages impair lung epithelial Na+ transport by an L-Arg-dependent mechanism.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1330-C1341,
1994
5.
Eaton, D. C.,
A. Becchetti,
H. Ma,
and
B. N. Ling.
Cellular regulation of amiloride blockable Na+ channels.
Biomed. Res. (Tokyo)
12:
31-35,
1991.
6.
Eaton, D. C.,
A. Becchetti,
H. Ma,
and
B. N. Ling.
Renal sodium channels: regulation and single channel properties.
Kidney Int.
48:
941-949,
1995[Medline].
7.
Feng, Z.,
R. B. Clark,
and
Y. Berthiaume.
Identification of nonselective cation channels in cultured adult rat alveolar type II cells.
Am. J. Respir. Cell Mol. Biol.
9:
248-254,
1993.
8.
Guidot, D. M.,
M. J. Repine,
B. M. Hybertson,
and
J. E Repine.
Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs.
Am. J. Physiol
269 (Lung Cell. Mol. Physiol. 13):
L2-L5,
1995
9.
Hummler, E.,
P. Barker,
J. Gatzy,
F. Beerman,
C. Verdumo,
A. Schmidt,
R. Boucher,
and
B. C. Rossier.
Early death due to defective neonatal lung liquid clearance in -ENaC-deficient mice.
Nat. Genet.
12:
325-328,
1996[Medline].
10.
Ichimori, K.,
H. Ishida,
M. Fukahori,
H. Nakazawa,
and
E. Burahami.
Practical nitric oxide measurement employing a nitric oxide-selective electrode.
Rev. Sci. Instrum.
65:
1-5,
1994.
11.
Ismailov, I. I.,
M. S. Awayda,
B. K. Berdiev,
J. K. Bubien,
J. E. Lucas,
C. M. Fuller,
and
D. J. Benos.
Triple barrel organization of EnaC, a cloned epithelial Na+ channel.
J. Biol. Chem.
271:
807-816,
1996
12.
Kase, H.
New inhibitors of protein kinases from microbial sources.
In: Biology of Actinomycetes 88: Proceedings of Seventh International Symposium on Biology of Actinomycetes, edited by Y. Okami,
T. Bappu,
and H. Ogawara. Tokyo: Japan Scientific Societies Press, 1988, p. 159-164.
13.
Kizer, N.,
X.-L. Guo,
and
K. Kruska.
Reconstitution of stretch activated cation channels by expression of the alpha subunit of the epithelial sodium channel cloned from osteoblasts.
Proc. Natl. Acad. Sci. USA
94:
1013-1018,
1997
14.
Koivisto, A.,
and
J. Nedergaard.
Modulation of calcium activated non-selective cation channel by nitric oxide in rat brown adipose tissue.
J. Physiol. (Lond.)
486:
59-65,
1995[Medline].
15.
Kokko, K. E.,
P. S. Matsumoto,
B. N. Ling,
and
D. C. Eaton.
Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron line (A6).
Am. J. Physiol.
267 (Cell Physiol. 36):
C1414-C1425,
1994
16.
Light, D. B.,
J. D. Corbin,
and
B. A. Stanton.
Dual ion channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase.
Nature
344:
336-339,
1990[Medline].
17.
Marunaka, Y.
Amiloride-blockable Ca2+-activated Na+-permeant channels in the fetal distal lung epithelium.
Pflügers Arch.
431:
748-756,
1996[Medline].
18.
Marunaka, Y.,
and
D. C. Eaton.
Chloride channels in the apical membrane of a distal nephron A6 cell line.
Am. J. Physiol.
258 (Cell Physiol. 27):
C352-C368,
1990
19.
Matalon, S.
Mechanisms and regulation of ion transport in adult mammalian alveolar type II pneumocytes.
Am. J. Physiol.
261 (Cell Physiol. 30):
C727-C738,
1991
20.
Matthay, M. A.,
H. G. Folkesson,
and
A. S. Verkman.
Salt and water transport across alveolar and distal airway epithelia in the adult lung.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L487-L503,
1996
21.
Moncada, S.,
R. M. J. Palmer,
and
E. A. Higgs.
Nitric oxide: physiology, pathophysiology, and pharmacology.
Pharmacol. Rev.
43:
109-142,
1991[Medline].
22.
O'Brodovich, H.,
V. Hannam,
M. Seear,
and
J. B. M. Mullen.
Amiloride impairs lung water clearance in newborn guinea pigs.
J. Appl. Physiol.
68:
1758-1762,
1990
23.
O'Brodovich, H.,
B. Rafii,
and
P. Perlon.
Arginine vasopressin and atrial natriuretic peptide do not alter ion transport by cultured fetal distal lung epithelium.
Pediatr. Res.
31:
318-322,
1992[Medline].
24.
Orser, B. A.,
L. Bertlik,
L. Fedorko,
and
H. O'Brodovich.
Cation selective channel in fetal alveolar type II epithelium.
Biochim. Biophys. Acta
1094:
19-26,
1991[Medline].
25.
Pepke-Zaba, J.,
T. W. Higgenbottam,
A. T. Dinh-Zuan,
D. Stone,
and
J. Wallwork.
Inhaled nitric oxide causes selective pulmonary vasodilatation in patients with pulmonary hypertension.
Lancet
338:
1173-1174,
1991[Medline].
26.
Rocha, A. S.,
and
L. H. Kudo.
Atrial peptide and cGMP effects on NaCl transport in inner medullary collecting duct.
Am. J. Physiol.
259 (Renal Fluid Electrolyte Physiol. 28):
F258-F268,
1990
27.
Rossiant, R.,
K. S. Falke,
F. Lopez,
K. Slama,
U. Pison,
and
W. M. Zapol.
Inhaled nitric oxide for the adult respiratory distress syndrome.
N. Engl. J. Med.
328:
399-405,
1993
28.
Scherrer, U.,
L. Vollenweider,
A. Delabays,
M. Savcic,
U. Eichenberger,
G. R. Kleger,
A. Fikrle,
P. E. Ballmer,
P. Nicod,
and
P. Bartsch.
Inhaled nitric oxide for high altitude pulmonary edema.
N. Engl. J. Med.
334:
624-629,
1996
29.
Stoos, B. A.,
N. H. Garcia,
and
J. L. Garvin.
Nitric oxide inhibits Na+ reabsorption in the isolated perfused cortical collecting duct.
J. Am. Soc. Nephrol.
6:
89-94,
1995[Abstract].
30.
Tohda, H.,
J. K. Foskett,
H. O'Brodovich,
and
Y. Marunaka.
Cl regulation of a Ca2+-activated nonselective cation channel in -agonist-treated fetal lung epithelium.
Am. J. Physiol.
266 (Cell Physiol. 35):
C104-C109,
1994
31.
Voilley, N. E.,
E. Lingueglia,
and
G. Champigny.
The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, autogenesis, and molecular cloning.
Proc. Natl. Acad. Sci. USA
91:
247-251,
1994
32.
Wahler, G. M.,
and
S. J. Dollinger.
Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase.
Am. J. Physiol.
268 (Cell Physiol. 37):
C45-C54,
1995
This article has been cited by other articles:
|
|
 |
|
  H. F. Bao, L. Liu, J. Self, B. J. Duke, R. Ueno, and D. C. Eaton A synthetic prostone activates apical chloride channels in A6 epithelial cells Am J Physiol Gastrointest Liver Physiol, August 1, 2008; 295(2): G234 - G251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Yu, H.-F. Bao, J. L. Self, D. C. Eaton, and M. N. Helms Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells Am J Physiol Renal Physiol, November 1, 2007; 293(5): F1666 - F1677. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Kaestle, C. A. Reich, N. Yin, H. Habazettl, J. Weimann, and W. M. Kuebler Nitric oxide-dependent inhibition of alveolar fluid clearance in hydrostatic lung edema Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L859 - L869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Song and S. Matalon Modulation of alveolar fluid clearance by reactive oxygen-nitrogen intermediates Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L855 - L858. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sobczak, A. Willing, K. Kusche, N. Bangel, and W.-M. Weber Amiloride-sensitive sodium absorption is different in vertebrates and invertebrates Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2007; 292(6): R2318 - R2327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. N. Helms, L. Yu, B. Malik, D. J. Kleinhenz, C. M. Hart, and D. C. Eaton Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia Am J Physiol Cell Physiol, September 1, 2005; 289(3): C717 - C726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-J. Chen, S. Seth, G. Yue, P. Kamat, R. W. Compans, D. Guidot, L. A. Brown, D. C. Eaton, and L. Jain Influenza virus inhibits ENaC and lung fluid clearance Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L366 - L373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chen, C. M. Fuller, T. R. Kleyman, and S. Matalon Mutations in the extracellular loop of {alpha}-rENaC alter sensitivity to amiloride and reactive species Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1202 - F1208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Matalon, K. M. Hardiman, L. Jain, D. C. Eaton, M. Kotlikoff, J. P. Eu, J. Sun, G. Meissner, and J. S. Stamler Regulation of ion channel structure and function by reactive oxygen-nitrogen species Am J Physiol Lung Cell Mol Physiol, December 1, 2003; 285(6): L1184 - L1189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Lazrak and S. Matalon cAMP-induced changes of apical membrane potentials of confluent H441 monolayers Am J Physiol Lung Cell Mol Physiol, August 1, 2003; 285(2): L443 - L450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Frank, J.-F. Pittet, H. Lee, M. Godzich, and M. A. Matthay High tidal volume ventilation induces NOS2 and impairs cAMP- dependent air space fluid clearance Am J Physiol Lung Cell Mol Physiol, May 1, 2003; 284(5): L791 - L798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tsubochi, S. Suzuki, H. Kubo, T. Ueno, T. Yoshimura, T. Suzuki, H. Sasano, and T. Kondo Early Changes in Alveolar Fluid Clearance by Nitric Oxide after Endotoxin Instillation in Rats Am. J. Respir. Crit. Care Med., January 15, 2003; 167(2): 205 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Matalon, A. Lazrak, L. Jain, and D. C. Eaton |
, Means ± SE of single-channel
currents.
on left and
right) from 16 cell-attached patch
experiments. Channel activity was measured as open probability
(Po) before and
2 min after addition of 100 µM GSNO to bath. Each
Po was calculated
from at least 3 min of consecutive recording. * GSNO decreased
Po by 43% from
control level, P < 0.001. C: summary of results (means ± SE;
